The present invention relates to a novel bovine VDJ recombination that is suitable for antigenization and use as a vaccine vector. Further modifications by antibody engineering techniques will also help generate immunoglobulin for targeting specific tissues or organs for therapeutic purposes. The unique composition of bovine germline D-genes provide additional opportunities for sustaining the capacity for antibody diversification in cattle essential for immunocompetence via selective breeding strategies that incorporate immunoglobulin gene markers. The novel gene elements, such as D-genes, are unique to cattle and, therefore, are useful for forensic analysis.
One of the most remarkable characteristics of the immune system is its ability to generate a large and diverse repertoire of antibody molecules that specifically recognize the native form of epitope(s) of potential pathological consequence in an attempt to protect the organism. Such a diversity that protects an organism from a variety of infectious pathogens and toxic substances has been exploited to produce high affinity receptors/antibody for use in immunodiagnostics, molecular probes and immunotherapy. For some applications, it is necessary to reduce antibody size to improve biodistribution, and reduce immunogenicity and side reactions arising from antibody effector functions. The Fv, consisting of associated VH and VL domains, constitutes the minimum functional antigen-binding fragment of a conventional antibody. Because of the inherent instability of VH-VL Fv heterodimers, single domain antibody, i.e., VH only, is expected to be stable because of its small size. This may be particularly so as homodimers of heavy (H)-chain alone occur naturally as antibody in camels. The murine VHs, unlike camel, are xe2x80x98stickyxe2x80x99 because of exposure of a large hydrophobic surface spanning 500-800 xc3x85xc2x0. In addition to poor solubility behaviour the murine VHs have an average CDR3H length of 9 amino acids in contrast to human (12 residues) and camels (14 residues). Thus, the potential antigen-binding surface of murine VH is smaller as compared to human antibodies of potential therapeutic significance. A Fv or single domain antibody with a longer CDR3H is, therefore, of potential interest from an antibody engineering perspective as it would potentially provide large antigen surface binding area.
The variable-region of immunoglobulin (Ig) bear V-region epitope capable of stimulating both humoral and cellular immune responses upon hetero-, allo or iso-immunization (Nisonoff, A. and Lamoyi, E. 1981. Implication of the presence of an internal image of the antigen in anti-idiotypic antibodies: possible application to vaccine production. Clin. 1 mm. Immunopath. 21:397-406; Zaghouani, H. et al. 1993. Presentation of a viral T cell epitope expressed in the CDR3 region of a self-immunoglobulin molecule. Science 259:224-227; Zaghouani, H. et al. 1993. Engineered immunoglobulin molecules as vehicles for T cell epitope. Intern. Rev. Immunol. 10:265-278). The V-region epitope of an Ig often correlates with the amino acid composition of the CDR regions. Since the CDR loops vary both in composition and size, these provide permissive sites for foreign (e.g., microbial origin) epitope insertion that would create an antigenized Ig of desired epitope. Prior research indeed demonstrates the ability to manipulate the entire CDRS of an immunoglobulin for generation of functional chimeric antibodies of interest (Morisson, Science 229:1202; Jones et al. 1986. Nature 321:522; Verhoeyen et al. 1988. Science. 239:1534; Riechmann et al. 1988. Nature 332:323). Antigenization of immunoglobulin is, however, hindered by shorter CDRs in immunoglobulins from mice and man that permit incorporation of T cell epitopes (linear) but not B cell epitopes (conformational) for use as vaccine vectors.
The inventors observed that approximately 9% of VDJ rearrangements encode functional bovine IgM antibody with a CDR3H length up to 61 amino acids with multiple cysteine residues (Saini, 1999). Such an exceptionally long CDR3H is the first ever to be documented in a species and is of significant interest to exploit its potential in engineering antibodies, either as Fv or single domain, of diagnostic and therapeutic significance. In addition to the long CDR3H, the configurational diversity from such a long primary sequence with multiple even numbered cysteine residues is expected to contribute to the immunoglobulin fold capable of binding to an infinite array of chemical structures.
Antigenization of immunoglobulin is hindered by shorter CDRs in immunoglobulins from mice and man that permit incorporation of T cell epitopes (linear) but not B cell epitopes (conformational) for use as vaccine vectors. The bovine VDJ cassette developed by the inventors provides the novel ability of the immunoglobulin molecule to incorporate both linear T cell epitopes (CDR1H and CDR2H) as well as conformational B cell epitopes (CDR3H). As a result, the germline encoded BF1H1 VDJ cassette isolated by the inventors from bovine fetal B cells is suitable for immunoglobulin antigenization with both B and T cell epitopes and use as vaccine vector. In addition, multiple epitopes can be incorporated and, thus, provide opportunity for development of multivalent vaccine. The isolation of expressed recombined VDJ genes capable of encoding CDR3H length  greater than 50 amino acids are the first ever to be documented in any species. The VDJ cassette isolated from cattle provides a natural source of immunoglobulin that is capable of incorporating multiple, both T and B cell epitopes, and is, therefore, most suitable as a vaccine vector across species by using species specific different isotypes, as or if required. The antigenized immunoglobulin incorporating both T and B cells of interest would be especially useful for development of oral vaccines for use in humans.
The long CDR3H in BF1H1 VDJ rearrangement originates from long germline D-genes so far known to exist in cattle only. The novel bovine germline D-genes provide additional opportunities for sustaining the capacity for antibody diversification in cattle essential for immunocompetence via selective breeding strategies that incorporate immunoglobulin gene markers. The novel gene elements, such as D-genes, are unique to cattle and, therefore, are of considerable use in forensic analysis as well.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.